The present invention relates to novel superactive insulin analogues and their use in pharmaceutical compositions for the treatment of diabetes.
Insulin is a hormone which has a key role in the regulation of growth and metabolism in vertebrates. Severe metabolic derangements occur in the absence of insulin resulting from the failure of many cells to utilize glucose and amino acids normally. The inability to metabolize glucose leads in man to diabetes mellitus, a complex chronic metabolic disorder in which there is abnormal carbohydrate, fat and protein metabolism. In its most fully expressed clinical form, diabetes mellitus is characterized by an absolute or relative deficiency of insulin or insulin activity and is associated with glucosuria, ketonuria, growth arrest, and negative nitrogen balance. These conditions can ultimately lead to death from acute metabolic acidosis caused by unrestrained oxidation of fatty acids or inanition which results from the lack of sufficient lipid reserves needed to generate ketone bodies. Inanition is defined as a condition characterized by marked weakness, extreme weight loss, and a decrease in metabolism resulting from prolonged and severe insufficiency of food. Dorland's Illustrated Medical Dictionary, 25th Edition.
The discovery and purification of insulin in the 1920's and its association with diabetes mellitus provided the means to intervene in the disease. See, e.g., Bliss, The Discovery of Insulin (1983), University of Chicago Press, Chicago, Ill. Today, insulin administration to diabetic patients is the primary therapeutic means for controlling the disease.
Insulin is a 6000 dalton polypeptide which is composed of two short peptide chains, termed A and B, which are linked to each other by invariant disulfide bridges. In almost all insulins studied, the A chain, which is 21 amino acids long, also contains an internal disulfide bridge. The B chain is 30 amino acids in length. Like many eukaryotic proteins, insulin is synthesized in a precursor form which is post-synthetically processed to the mature two polypeptide chain active hormone.
The immediate precursor of insulin is proinsulin, a single chain polypeptide composed of the B and A chains linked to a connecting peptide of approximately 31 amino acids, termed the C-peptide, by adjacent pairs of basic residues. The order of the three peptides in the proinsulin molecule is NH.sub.2 -B chain-Arg-Arg-C-peptide-Lys-Arg-A chain-COOH. The translation product of insulin mRNA, however, is preproinsulin which is proinsulin that contains at its NH.sub.2 terminus a 24 amino acid largely hydrophobic signal peptide characteristic of proteins that are either transported through or inserted into cellular membranes.
Preproinsulin is synthesized in pancreatic beta cells located within the islets of Langerhans which are dispersed throughout the pancreas. Removal of the signal peptide occurs in the rough endoplasmic reticulum with the resulting fully folded oxidized proinsulin being transported to the Golgi apparatus for packaging into secretion granules. The folded proinsulin is stabilized by disulfide bonds. During maturation of the secretion granules, the folded proinsulin molecule is cleaved by specific proteases at the paired basic residues to liberate insulin and the C-peptide.
As discussed above, therapy for diabetes mellitus includes administration of controlled amounts of insulin to the diabetic patient. The insulin so administrated has, for the most part, been obtained from animal pancreases, notably bovine and porcine. Bovine and porcine insulins function to maintain hormonal homeostasis in the same way as human insulin with about the same potency but, because they are foreign proteins, can elicit an immunological response which diminishes their usefulness. More recently, human insulin, generated by recombinant DNA techniques, has been added to the therapeutic armamentarium. The use of human insulin, produced by recombinant DNA or other techniques, is not likely to produce the adverse immunological problems attendant the use of animal insulins. Even with the availability of natural human insulin, however, administration of the hormone to diabetics has not been always sufficient to restore normal metabolism. There is thus a need for alternative insulins with better activity or other means of therapy for diabetes.
Familial hyperproinsulinemia is a genetic disorder characterized by a marked increase in serum proinsulin-like molecules. Three families with this rare disorder have been identified. In two of the families a structurally abnormal proinsulin-like molecule was seen. The genetic defect was identified as a mutation causing an amino acid substitution in proinsulin which results in incomplete cleavage of proinsulin by the proteases which form insulin.
The affected members of the third family produced a proinsulin-like molecule of about the same size as proinsulin that behaved like the normal prohormone in receptor binding assays. Sequence analysis of cloned insulin genes obtained from two affected members from this third family revealed a single coding mutation which substituted an aspartic acid for histidine in the proinsulin molecule at a position which corresponds to position 10 of the B chain of insulin. Chan et al., Proc. Natl. Acad. Sci. (1987), vol. 84, pp. 2194-2197. The mutation was believed to inhibit further processing of proinsulin to insulin, thus resulting in the accumulation of the mutant proinsulin. The precise way the mutation inhibits further processing is not currently known. A human insulin analogue, [10-Aspartic acid-B] human insulin, which corresponds to this mutant proinsulin, has now been synthesized and has been shown to have greater potency than natural insulins.
It has also been found that the elements of the carboxyl terminal of the B chain of insulin appear also to influence the biological activity of insulin. Specifically, the B25 site appears also to play a role in the potency of insulin analogues.
Elimination of the C-terminal pentapeptide sequence of the B-chain of insulin, and amidation of the carboxyl group of the newly-formed C-terminus, Phe B25, results in an analogue, des-pentapeptide(B26-B30)-[Phe.sup.B25 -.alpha.-carboxamide]insulin, which has been shown to display comparable potency with the natural hormone. See Nakagawa et al., J. Biol. Chem., 261:7332-41(1986); Cosmatos et al., Int. J. Pept. Prot. Res., 14:457-71 (1979); Casareto et al., Biol. Chem. Hoppe-Seyler, 368:709-16 (1987). Substitution of Phe B25 with several-other amino acid residues, as well as various modifications of the B26-B30 segment of these substituted insulins, led to analogues varying in potency from almost total inactivity to potency higher than natural insulin. Nakagawa et al., J. Biol. Chem., 261, supra; Casareto et al., supra; Nakagawa et al., J. Biol. Chem., 262:10254-58 (1987). Among these, des-pentapeptide(B26-B30)-[Tyr.sup.B25 -.alpha.-carboxamide]insulin and its His.sup.B25 analogue display potency about 270-300% greater than insulin. Based upon these studies, it has been suggested that the B25 amino acid residue of insulin interacts with the receptor, thereby initiating conformational changes in as yet undefined areas of the insulin molecule which are involved in hormone-receptor binding. The B25-receptor interaction may be modulated in a positive or negative manner by the C-terminal B-chain domain, depending on the nature of the modifications to the B25 residue and the extent to which the B chain C-terminal domain has been altered. Nakagawa et al., J. Biol. Chem., 261, supra; Casareto, supra; Nakagawa et al., J. Biol. Chem., 262, supra.
Another human insulin analogue, des-pentapeptide (B26-B30)-[Asp.sup.B10, Tyr.sup.B25 -.alpha.-carboxamide] insulin has also been synthesized and shown to have greater potency than natural insulins.